The present invention relates to a human/mouse chimeric antibody comprising variable region (V region) of a mouse monoclonal antibody against human tissue factor (TF) and constant region (C region) of a human antibody; a humanized antibody in which complementarity determining regions (CDRs) of light chain (L chain) V region and heavy chain (H chain) V region of a mouse monoclonal antibody against human TF have been grafted onto a human antibody; the L chain and H chain of said antibody; and a V region fragment constituting the L chain or H chain of said antibody. The present invention also relates to a process of production of a humanized antibody against human TF.
The present invention also relates to DNA encoding the above antibody, specifically a V region fragment thereof, and DNA encoding an L chain or H chain that contains a V region. The present invention also relates to a recombinant vector comprising said DNA, and a host transformed with said vector.
The present invention also relates to a process of preparing a chimeric antibody and a humanized antibody against human TF. The present invention also relates to a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation (DIC) syndrome comprising as an active ingredient a humanized antibody against human TF.
Tissue factor (TF), a receptor of the coagulation factor VII expressed on the cell surface, plays an indispensable role in the activation of coagulation factors IX and X through the formation of a complex with the coagulation factor VII, and has been defined as a practical initiating factor of blood coagulation reactions.
TF is known to be expressed in fibroblasts, smooth muscle cells, etc. that constitute the blood vessel, and to play a hemostatic function by activating the coagulation system at the time of blood vessel injury.
DIC is a disease in which the activation of the coagulation system in a blood vessel leads to systemic multiple occurrence of blood clots, mainly in the microvasculature. It is not uncommon that the reduction of platelets and coagulation factors due to consumption leads to bleeding which is the opposite phenomenon to blood clotting. The multiple microthrombi can cause deficient microcirculation in the mayor organs, which, once developed, leads to irreversible functional deficiency and to bad prognosis of DIC, and in this sense DIC is considered an important disease.
The incidence of underlying diseases estimated from the 1990 and 1992 research reports by the Ministry of Health and Welfare Specified Diseases Blood Coagulation Disorders Survey and Study Group is: hematological malignancies, about 30%; solid tumors about, 20%; infections, about 15%; obstetric diseases, about 10%; hepatic diseases about, 6%; shocks, about 5%; and cardiovascular diseases, about 3%. The incidence of DIC is as high as about 15% in leukemia and about 6 to 7% in malignant lymphoma, and about 3% in solid tumors.
DIC develops accompanied by various diseases mentioned above, but the causative agent thereof is the same, which is TF. Thus, the onset mechanism of DIC is believed to be: abnormally high formation and/or expression of TF in cancer cells in acute leukemia, malignant lymphoma, and solid tumors; the enhanced formation and/or expression of TF in monocytes and/or endothelial cells in infections (in particular, sepsis caused by Gram-negative bacilli); TF influx into the blood from the necrotized liver tissue in fulminant hepatitis; TF expression on the lumina of the blood vessel in aortic aneurysm, cardiac aneurysm, and giant hemangioma; and also TF influx into the blood in obstetric diseases (amniotic fluid embolism and abruptio placentae) and surgeries, injuries, and burns.
The treatment of the original (underlying) diseases is of utmost concern, which, however, is not easy in practical terms.
As a current method of treating DIC, anticoagulant therapy and substitution therapy are in use. Heparin preparations (fractionated heparin, low molecular weight heparin) are mainly used for anti-coagulant therapy. Synthetic protease inhibitors (gabexate mesilate, nafamostat mesilate) and concentrated plasma (anti-thrombin III, activated protein C preparations) are also used. As a substitution therapy, there are platelet concentrates, fresh frozen plasmas (supply of fibrinogen), washed red blood cells, and the like.
However, the current therapeutic agents are not satisfactory in terms of efficacy and side effects, and in most cases complete withdrawal from DIC is impossible. Therefore, there is a need for the use of drugs having high therapeutic effects and low side effects.
On the other hand, as new attempts in DIC treatments there can be mentioned thrombomodulin preparations, hirudin, and anti-PAF agents, Tissue factor pathway inhibitor (TFPI). FXa-selective inhibitors are attracting attention as orally administrable anticoagulant and/or antithrombotic agents. Also as an agent that neutralizes the activity of TF, WO 88/07543 discloses mouse anti-human TF monoclonal antibody, and WO 96/40921 discloses humanized anti-human TF antibody.
Mouse anti-human TF monoclonal antibodies are expected to make a safe and effective therapeutic agent in that it does not exhibit a symptom of bleeding associated with main efficacy in DIC. However, mouse monoclonal antibodies are highly immunogenic (sometimes referred to as xe2x80x9cantigenicxe2x80x9d), and thus the medical therapeutic value of mouse antibodies in humans is limited. For example, the half life of mouse antibodies in humans is relatively short and therefore they cannot fully exhibit their anticipated effects. Furthermore, human anti-mouse antibody (HAMA) that develops in response to the mouse antibody administrated causes immunological reactions that are unfavorable and dangerous to patients. Thus, mouse monoclonal antibodies cannot be repeatedly administered to humans.
In order to solve these problems, methods have been developed that intend to reduce the immunogenicity of antibodies derived from non-humans, such as (monoclonal antibodies derived from) mice. One of them is a method of making chimeric antibody in which a variable region (V region) of the antibody is derived from mouse monoclonal antibody, and a constant region (C region) thereof is derived from a suitable human antibody.
Since the chimeric antibody obtained contains variable region of an original mouse antibody in the complete form, it is expected to bind to an antigen with the identical affinity as that of the original mouse antibody. Furthermore, in chimeric antibody the ratio of the amino acid sequences derived from non-humans is substantially reduced, and thereby it is expected to have a reduced immunogenicity compared to the original mouse antibody. However, it is still possible for an immunological response to the mouse variable region to arise (LoBuglio, A. F. et al., Proc. Natl. Acad. Sci. USA, 86: 4220-4224, 1989).
A second method of reducing the immunogenicity of mouse antibody is, though much more complicated, expected to drastically reduce the potential immunogenicity of mouse antibody. In this method, the complementarity determining region (CDR) alone of a mouse antibody is grafted onto a human variable region to make a xe2x80x9creshapedxe2x80x9d human variable region. As desired, however, some amino acid sequences of framework regions (FRs) supporting the CDRs may be grafted from the variable region of a mouse antibody onto the human variable region in order to obtain the closest possible approximation of the original mouse antibody structure. Then, the humanized reshaped human variable region is ligated to the human constant region. In the finally reshaped humanized antibody, portions derived from non-human amino acid sequences are only the CDRs and a small portion of the FRs. The CDRs comprise hypervariable amino acid sequences and they do not show species-specific sequences.
For humanized antibody, see also Riechmann, L. et al., Nature 332: 323-327, 1988; Verhoeye, M. et al., Scienece 239: 1534-1536, 1998; Kettleborough, C. A. et al., Protein Engng., 4: 773-783, 1991; Maeda, H., Human Antibodies and hybridoma, 2: 124-134, 1991; Gorman, S. D. et al, Proc. Natl. Acad. Sci. USA, 88: 4181-4185, 1991; Tempest, P R., Bio/Technology, 9: 226-271, 1991; Co, M. S. et al., Proc. Natl. Acad. Sci. USA 88: 2869-2873, 1991; Cater, P. et al., Proc. Natl. Acad. Sci. USA, 89: 4285-4289, 1992; Co, M. S. et al., J. Immunol., 148: 1149-1154, 1992; and Sato, K. et al., Cancer Res., 53: 851-856, 1993.
In the conventional humanization technology, part of the framework region (FR) includes an amino acid sequence that was grafted from the variable region of a mouse antibody to the human variable region. Thus, when it is administered as a therapeutic agent in humans, there is a risk that antibodies are formed against a site having an amino acid sequence not present in humans, though it is merely one to a few amino acids. In order to circumvent the risk, a third humanization technology was devised. Thus, the method involves, for four FRs (FR1-4) required to support the three dimensional structure of three CDRs, the substitution of the FR of a human antibody having a high homology with the FR of the mouse antibody present in the database using one FR as a unit. In this case, several FRs are selected from human antibodies present in the database, and are sequentially shuffled to prepare a humanized antibody having a high activity.
By so doing, it is possible to construct humanized antibodies in which all the FRs except the CDRs in the variable region have amino acid sequences derived from human antibody. Thus, the humanized antibody carrying the mouse CDR should no longer have immunogenicity more potent than a human antibody containing the human CDR.
Although humanized antibody is expected to be useful for the purpose of treatment, as mentioned above, there is no fixed process present which is universally applicable to any antibody in the method of producing humanized antibody, and thereby various contrivances are required to construct a humanized antibody that exhibits a sufficient binding activity and neutralizing activity to a specific antigen (see, for example, Sato, K. et al., Cancer Res., 53: 851-856, 1993).
It is an object of the present invention to provide a human/mouse chimeric antibody comprising the variable region (V region) of a mouse monoclonal antibody against human tissue factor (TF) and the constant region (C region) of a human antibody, a humanized antibody in which the complementarity determining regions (CDRs) of the light chain (L chain) V region and the heavy chain (H chain) V region of a mouse monoclonal antibody against human TF have been grafted onto a human antibody, the L chain and H chain of said antibody, and a V region fragment constituting the L chain or H chain of said antibody. It is a further object of the present invention to provide a process of making a humanized antibody against human TF.
It is a further object of the present invention to provide DNA encoding the above antibody, specifically a V region fragment thereof, and DNA encoding an L chain or H chain that contains a V region. It is a further object of the present invention to provide a recombinant DNA vector comprising said DNA, and a host transformed with said vector. It is a further object of the present invention to provide a pharmaceutical composition and a therapeutic agent for disseminated intravascular coagulation syndrome (DIC) comprising as an active ingredient a humanized antibody against human TF.
After intensive study to solve the above problems, the inventors of the present invention have successfully obtained an antibody in which immunogenicity in humans of the mouse monoclonal antibody against human TF is reduced, and also developed a process of making a novel humanized antibody, and thereby have completed the present invention.
Thus, the present invention relates to a chimeric H chain comprising the H chain C region of a human antibody and a fragment of the H chain V region of a mouse monoclonal antibody against human TF. As the H chain V region, there can be mentioned one that comprises an amino acid sequence as set forth in SEQ ID NO: 156, and as the C region, there can be mentioned one that is derived from the Cxcex34 region.
Furthermore, the present invention relates to a chimeric L chain comprising the L chain C region of a human antibody and a fragment of the L chain V region of a mouse monoclonal antibody against human TF. As the L chain V region, there can be mentioned one that comprises an amino acid sequence as set forth in SEQ ID NO: 162, and as the L chain C region, there can be mentioned one that is derived from the Cxcexa region.
Furthermore, the present invention relates to a human/mouse chimeric monoclonal antibody against human TF, said antibody comprising the above chimeric H chain and chimeric L chain.
The present invention also relates to a H chain V region fragment of a humanized antibody, said fragment comprising the framework regions (FRs) 1-4 of the H chain V region of a human antibody and the complementarity determining regions (CDRs) 1-3 of the H chain V region of a mouse monoclonal antibody against human TF. As the CDR -3, there can be mentioned one that includes the amino acid sequence as set forth in SEQ ID NO: 133-135, respectively. As the FR1 of the H chain V region of a human antibody, there can be mentioned the human antibody FR1 that has a homology of 40% or greater with the FR1 of the H chain V region of a mouse antibody; as the FR2, there can be mentioned the human antibody FR2 that has a homology of 40% or greater with the FR2 of the H chain V region of a mouse antibody; as the FR3, there can be mentioned the human antibody FR3 that has a homology of 40% or greater with the FR3 of the H chain V region of a mouse antibody; and as the FR4, there can be mentioned the human antibody FR4 that has a homology of 40% or greater with the FR4 of the H chain V region of a mouse antibody.
Preferably, as the FR1 of the H chain V region of a human antibody, there can be mentioned the human antibody FR1 that has a homology of 50% or greater with the FR1 of the H chain V region of a mouse antibody; as the FR2, there can be mentioned the human antibody FR2 that has a homology of 70% or greater with the FR2 of the H chain V region of a mouse antibody; as the FR3, there can be mentioned the human antibody FR3 that has a homology of 65% or greater with the FR3 of the H chain V region of a mouse antibody; and as the FR4, there can be mentioned the human antibody FR4 that has a homology of 80% or greater with the FR4 of the H chain V region of a mouse antibody. As specific examples, as the FR1 of the H chain V region of a human antibody, there can be mentioned the human antibody L39130; as the FR2, there can be mentioned the human antibody L39130, the human antibody P01742, and the human antibody Z80844; as the FR3, there can be mentioned the human antibody L39130, the human antibody Z34963, the human antibody P01825, the human antibody M62723, the human antibody Z80844, the human antibody L04345, the human antibody S78322, the human antibody Z26827, the human antibody U95239, and the human antibody L03147; and as the FR4, there can be mentioned the human antibody L39130.
As preferred examples, as the FR1 of the H chain V region of a human antibody, there can be mentioned the human antibody L39130; as the FR2, there can be mentioned the human antibody L39130 and the human antibody Z80844; as the FR3, there can be mentioned the human antibody Z34963, the human antibody M62723, and the human antibody U95239; and as the FR4, there can be mentioned the human antibody L39130. As more preferred examples, as the FR1 of the H chain V region of a human antibody, there can be mentioned the human antibody L39130; as the FR2, there can be mentioned the human antibody L39130; as the FR3, there can be mentioned the human antibody Z34963 and the human antibody U95239; and as the FR4, there can be mentioned the human antibody L39130.
Furthermore, as used herein, numbers in the framework regions are based on definition by Kabat (Kabat, E. A. et al., US Dept. Health and Services, US Government Printing Offices, 1991).
The present invention also relates to a H chain V region fragment of a humanized antibody, said fragment comprising either of the amino acid sequences as set forth in SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64, 70, 72, 76, 78, 82, and 84.
The present invention also relates to an L chain V region fragment of a humanized antibody, said fragment comprising the FRs 1-4 of the L chain V region of a human antibody and the CDRs 1-3 of the L chain V region of a mouse monoclonal antibody against human TF. As the CDRs 1-3, there can be mentioned one that includes the amino acid sequence as set forth in SEQ ID NO: 136-138, respectively. As the FR1 of the L chain V region of a human antibody, there can be mentioned one that has a homology of 40% or greater with the FR1 of the L chain V region of a mouse antibody; as the FR2, there can be mentioned the human antibody FR2 that has a homology of 40% or greater with the FR2 of the L chain V region of a mouse antibody; as the FR3, there can be mentioned the human antibody FR3 that has a homology of 40% or greater with the FR3 of the L chain V region of a mouse antibody; and as the FR4, there can be mentioned the human antibody FR4 that has a homology of 40% or greater with the FR4 of the L chain V region of a mouse antibody.
Preferably, as the FR1 of the L chain V region of a human antibody, there can be mentioned the human antibody FR1 that has a homology of 75% or greater with the FR1 of the L chain V region of a mouse antibody; as the FR2, there can be mentioned the human antibody FR2 that has a homology of 80% or greater with the FR2 of the L chain V region of a mouse antibody; as the FR3, there can be mentioned the human antibody FR3 that has a homology of 70% or greater with the FR3 of the L chain V region of a mouse antibody; and as the FR4, there can be mentioned the human antibody FR4 that has a homology of 80% or greater with the FR4 of the L chain V region of a mouse antibody. As specific examples, as the FR1 of the L chain V region of a human antibody, there can be mentioned the human antibody Z37332; as the FR2, there can be mentioned the human antibody Z37332 and the human antibody X93625; as the FR3, there can be mentioned the human antibody Z37332, the human antibody S68699, and the human antibody P01607; and as the FR4, there can be mentioned the human antibody Z37332. As more preferred examples, as the FR1 of the L chain V region of a human antibody, there can be mentioned the human antibody Z37332; as the FR2, there can be mentioned the human antibody X93625; as the FR3, there can be mentioned the human antibody S68699; and as the FR4, there can be mentioned the human antibody Z37332.
Furthermore, as used herein, numbers in the framework regions are based on definition by Kabat (Kabat, E. A. et al., US Dept. Health and Services, US Government Printing Offices, 1991).
The present invention also relates to an L chain V region fragment of a humanized antibody, said fragment comprising the amino acid sequences as set forth in SEQ ID NO: 93, 99, 101, 107, and 109.
The present invention also relates to the H chain of a humanized antibody against human TF, said chain comprising a H chain V region fragment of the above humanized antibody and a H chain C region fragment of a human antibody. There can be mentioned the Cxcex34 region as the C region; as the FRs 1-4 derived from a human antibody, there can be mentioned those each derived from the human antibody L39130 (FR1), the human antibody L39130 (FR2), the human antibody Z34963 (FR3) or the human antibody U95239 (FR3), the human antibody L39130 (FR4); and as the CDRs 1-3, there can be mentioned those each derived from the amino acid sequence as set forth in SEQ ID NO: 133-135, respectively.
The present invention also relates to the L chain of a humanized antibody against human TF, said chain comprising an L chain V region fragment of the above humanized antibody and an L chain C region fragment of a human antibody. There can be mentioned the Cxcexa region as the C region; as the FRs 1-4 derived from a human antibody, there can be mentioned those each derived from the human antibody Z37332 (FR1), the human antibody X93625 (FR2), the human antibody S68699 (FR3), and the human antibody Z37332 (FR4); and as the CDRs 1-3, there can be mentioned those each derived from the amino acid sequence as set forth in SEQ ID NO: 136-138, respectively.
The present invention also relates to a humanized antibody against human TF, said antibody comprising the L chain and H chain of the above humanized antibody.
The present invention also relates to a process of making a humanized antibody against human TF. The process of humanization relates to the method of selecting the FRs 1-4 supporting the structure of the CDRs 1-3 that are the antigen recognition site for the H chain or L chain. Thus, the present invention relates to the method of selecting some of FRs of a human antibody having a high homology with the FR of a mouse antibody with each FR as a unit, and generating a humanized antibody having the desired activity by a sequential reshuffling of the FR.
More specifically, one example of a process of preparing a natural humanized antibody that has a complementarity determining region (CDR) derived from non-humans and a framework region (FR) derived from a natural human antibody and that has a reduced immunogenicity, said method comprising the steps of:
(1) preparing a non-human monoclonal antibody responsive to the antigen of interest;
(2) preparing some of human antibodies having a high homology with the amino acid sequence of the FR in the monoclonal antibody of the above (1);
(3) replacing the four FRs of one human antibody in the above (2) with the corresponding FRs of the non-human monoclonal antibody of the above (1) to generate the first humanized antibody;
(4) determining the ability of the humanized antibody generated in the above (3) to bind to the antigen or to neutralize the biological activity of the antigen;
(5) replacing one to three FRs of the humanized antibody generated in the above (3) with the corresponding FRs of a human antibody that is different from the one used in (3) among the human antibodies prepared in (2) to generate the second humanized antibody;
(6) comparing the ability of the second humanized antibody generated in the above (5) and the first humanized antibody generated in the above (3) for the ability to bind to the antigen or to neutralize the biological activity of the antigen thereby to select a humanized antibody that has a favorable activity;
(7) performing the above steps of (3) to (6) for the humanized antibody selected in the above (6); and
(8) repeating the above steps of (3) to (6) until a humanized antibody having an equivalent activity to the non-human monoclonal antibody in the above (1) is obtained.
Once a humanized antibody having a certain degree of activity of neutralizing human TF is obtained, further homology search is carried out for a specific FR in the H chain and L chain V region so that a human antibody having a high homology can be selected. By adding the thus obtained human antibody to a group of some human antibodies in the above step (2) and further repeating the steps of (3) to (6), a humanized antibody having the desired activity can be obtained.
The present invention also relates to DNA encoding a H chain V region fragment or an L chain V region fragment of a mouse monoclonal antibody against human TF. As the amino acid sequence and coding DNA of the H chain V region fragment or L chain V region fragment, there can be mentioned one that includes the nucleotide sequence as set forth in SEQ ID NO: 9 or 15, respectively.
The present invention also relates to DNA encoding the above chimeric H chain or chimeric L chain. As DNA encoding said H chain, there can be mentioned one that includes the nucleotide sequence as set forth in SEQ ID NO: 9, and as DNA encoding said L chain, there can be mentioned one that includes the nucleotide sequence as set forth in SEQ ID NO: 15.
The present invention also relates to DNA encoding a H chain V region fragment or L chain V region fragment of the above humanized antibody. As the DNA encoding the H chain V region fragment, there can be mentioned one that includes either of the nucleotide sequences as set forth in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59, 63, 69, 71, 75, 77, 81, or 83, and as the DNA encoding the L chain V region fragment, there can be mentioned one that includes either of the nucleotide sequences as set forth in SEQ ID NO: 92, 98, 100, 106, or 108.
The present invention also relates to DNA encoding the H chain of a humanized antibody.
The present invention also relates to the H chain DNA of a humanized antibody, said DNA comprising DNA encoding either of the amino acid sequences as set forth in SEQ ID NO: 30, 40, 42, 50, 52, 58, 60, 64, 70, 72, 76, 78, 82, or 84. As said DNA, there can be mentioned one that includes either of the nucleotide sequences as set forth in SEQ ID NO: 29, 39, 41, 49, 51, 57, 59, 63, 69, 71, 75, 77, 81, or 83.
The present invention also relates to DNA encoding the L chain of the above humanized antibody.
The present invention also is the L chain DNA of a humanized antibody, said DNA comprising DNA encoding the amino acid sequences as set forth in SEQ ID NO: 93, 99, 101, 107, or 109. As said DNA, there can be mentioned one that includes either of the nucleotide sequences as set forth in SEQ ID NO: 92, 98, 100, 106, or 108.
The present invention also relates to a recombinant DNA vector containing either of the DNA described above.
The present invention also relates to a transforming transformed with a recombinant DNA vector described above.
The present invention also relates to a process of generating a chimeric antibody or a humanized antibody against human TF, said method comprising culturing the above transformant and obtaining a chimeric antibody or a humanized antibody against human TF from the culture harvested.
The present invention also relates to a pharmaceutical composition and a therapeutic agent for DIC comprising the above humanized antibody as an active ingredient.